Evaluation of an IgM-ELISA test for the diagnosis of human leptospirosis.

Leptospirosis is a zoonosis with a worldwide distribution very common in most countries. In Italy this acute febrile illness is more frequent in the Northern than in the Southern regions. In the period 1994-1996, the number of cases of Leptospirosis in Sicily was lower with respect to the northern-central regions (7.2% and 73.4% respectively). Between January 1990 and December 1999, a total of 9 leptospirosis cases were observed in the Regional Centre for Leptospirosis of Palermo. The patients were all males (age between 22 and 59 years) and their occupations varied. Laboratory diagnosis is performed by the classical microagglutination microscopical (MAT) but this test is very complex and time-consuming. This study compared the classical MAT with ELISA IgM by using 19 serum samples from 9 patients with confirmed leptospirosis. We also tested 23 serum samples from blood-donors and 29 serum samples from patients with other infectious diseases. By the MAT and the PanBio IgM ELISA all sera from patients were found to be positive. Our results indicate that MAT represents the test with the highest degree of specificity (100%), but ELISA is simpler to perform, considering the favourable degree of sensitivity (100%) and specificity (95.9%).

The acute phase response in Sicilian patients with boutonneuse fever admitted to hospitals in Palermo, 1992-1997.

OBJECTIVES: To study the modifications of some components of the acute phase response (APR) in Sicilian patients with boutonneuse fever (BF) caused by Rickettsia conorii. METHODS: Sera from 500 Sicilian patients with confirmed BF were studied at the time of diagnosis and every week after treatment, and after recovery for the presence of various inflammatory mediators. Tumour necrosis factor alpha (TNFalpha), interleukin(IL)-6, IL-1alpha, IL-8, soluble TNF receptors (sTNF-R) and sIL-6R were assayed by commercially ELISA kits. C3, C4, factor B, C-reactive protein (CRP), fibrinogen, ceruloplasmin (Cp) and alpha(1)-antitrypsin (AAT) were assayed by a rate nephelometry. RESULTS: Interferon gamma (IFNgamma), IL-6, TNFalpha, and IL-10 cytokines were significantly modified, whereas IL-1 and IL-8 were not detectable in the blood in any phase of infection. sTNF-RI, sTNF-RII and sIL-6 were significantly increased in the first 2 weeks of infection, but sTNF-R levels were not related to the plasma levels of TNFalpha, whereas sIL-6 was directly related to serum IL-6 concentrations. C3, C4, factor B and CRP were significantly increased in the first 2 weeks of infection, but afterwards returned to the normal range, even though CRP was still high in the third week and C3 persisted high after the fourth week. Fibrinogen was high only in the first week in relation to the injury to the endothelial cells (ECs). The anti-inflammatory proteins, Cp and AAT, were extremely high in the first 2 weeks of infection acting as a buffer of APR activation. CONCLUSIONS: These results suggest that R. conorii is able to elicit, after invasion and proliferation in the ECs, the activation of APR. Further work is required to establish if active inhibitory mechanisms are operating during APR, or if there is a spontaneous decay in the initiation events.

Differential up-regulation of circulating soluble selectins and endothelial adhesion molecules in Sicilian patients with Boutonneuse fever.

In 150 patients with Boutonneuse fever (BF), caused by Rickettsia conorii, we studied the plasma levels of soluble L-selectin (sL-selectin), vascular cell adhesion molecule-1 (sVCAM-1), intercellular adhesion molecule-1 (sICAM-1) and E-selectin (sE-selectin) in various phases of disease to clarify their role in disease evolution. Results indicate that during the acute phase of BF there is a significant increase in the serum levels of sL-selectin, sE-selectin, sVCAM-1 and sICAM-1. sL-selectin and sVCAM-1 returned to normal levels in the third week of disease, whereas sE-selectin and sICAM-1 persisted at significantly high levels even after the third week. The secretion of these soluble CAMs in BF is mainly the result of leucocyte expression and endothelial cell activation, but secretion also appears to mediate anti-inflammatory activities, moderating leucocyte adhesion and reducing in particular lymphocyte and monocyte infiltration. Only sL-selectin serum levels were found to correlate with the acute phase of infection characterized by fever.

Modifications of general parameters of immune activation in the sera of Sicilian patients with Boutonneuse fever.

The serum levels of beta2-microglobulin (beta2-M), soluble HLA class I antigen (sHLA-I), soluble CD4 (sCD4) and CD8 (sCD8) were studied in 98 Sicilian patients with Boutonneuse fever (BF). In different stages of infection all markers were significantly increased in sera from Sicilian patients with acute BF compared with healthy controls. sCD8 and sHLA-I reached the peak in the second week after the onset of symptoms, whereas sCD4 and beta2-M reached the peak in the first week. Afterwards sCD8 decreased to the levels of controls within the third week, the other parameters decreased later and were unmodified until the third week of infection. Significant correlations were found between sCD4 and sCD8 and the sIL-2R, as well as between serum levels of beta2-M and sCD8. The reduction of CD3+ and CD4+ and the increase of CD8+ T cells in the blood indicate that these cells are involved in the response to rickettsia, and their activation might be in part responsible for the release of sCD4 and sCD8. Our data suggest that these soluble markers, indexes of immune activation of T cells both in the circulation and the affected tissues, may be used in monitoring BF evolution.

Depression of CD4 T cell subsets and alteration in cytokine profile in boutonneuse fever.

Interferon (IFN)-gamma, interleukin (IL)-10, IL-6, and tumor necrosis factor (TNF)-alpha were significantly increased in sera from Sicilian patients with acute boutonneuse fever (BF) compared with those of healthy controls. IFN-gamma levels dropped sharply within the second week after infection. IL-6, IL-10, and TNF-alpha levels gradually declined; in convalescent patients only were they in the normal range. In contrast, peripheral blood mononuclear cells (PBMC) stimulated in vitro with phytohemagglutinin (PHA) produced low levels of IL-10 and IFN-gamma in acute BF that were compatible with the reduction in the levels of CD4+, CD4+/CD45RO+, and CD4+/CD45RA+ cells. In vitro production of TNF-alpha and IL-6 from PBMC stimulated with PHA was not significantly modified during the various phases of the infection compared with control PBMC, which could be due to the persistence of high levels of CD14+ monocytes compensating for the decrease in CD20+ B cells.

In vivo and in vitro cytokine profiles and mononuclear cell subsets in Sicilian patients with active visceral leishmaniasis.

Sera from Sicilian patients with confirmed visceral leishmaniasis (Leishmania donovani infantum) were analysed at the moment of the diagnosis, during the course of the disease and after clinical recovery, for the concentration of IL-10, IFN-gamma, IL-4 and IL-2. The results show high concentrations of IL-10 and IFN-gamma in the sera at the beginning of infection that return to the normal range following successful chemotherapy. By contrast, PBMC stimulated in vitro with Ag and mitogen produced low levels of IL-10 and IFN-gamma when collected at the time of the diagnosis and normal levels when assayed after recovery. IL-2 was undetected in the sera and was significantly reduced in the supernatants of actively infected patients, returning to the normal level after recovery. IL-4 was absent in the sera and in high concentrations in the supernatants in all the phases of the disease. The levels of CD4+ and CD8+ T cells were within the normal range, but acute VL patients had markedly reduced levels of memory T cells (CD3+/CD45RO+) compared with healthy controls. These cells returned to the normal levels following successful chemotherapy. T cells are strongly activated in acute VL patients as indicated by the elevated number of CD3+ HLA-DR+ and by the increase in HLA-DR antigen on these cells. There was a significant reduction in the cell membrane DR antigen of the monocytes (CD 14+) during the acute phase of the disease, but it returned to the normal range after clinical recovery. These findings therefore suggest that in Sicilian patients with active VL the cytokine profile is not clearly characterized by Th2 phenotype as in mice, and both Th1-like and Th2-like cells appear to proliferate and to be activated. Furthermore, IL-10, rather than IL-4, could play an important part in the inhibition of IFN-gamma-induced macrophage activation and could reflect the levels of HLA-DR antigen expressed by the monocytes.

Evaluation of serum levels of soluble CD4, CD8 and beta 2-microglobulin in visceral human leishmaniasis.

The levels of soluble CD4 (sCD4), sCD8 and beta 2-microglobulin (beta 2-M) were measured in sera from patients with visceral leishmaniasis during the course of infection. Levels of sCD4, sCD8 and beta 2-M were raised significantly above levels in normal sera and returned to the normal range after recovery. The decrease in the levels of sCD8 was related to a reduction of anaemia, leukopenia and thrombocytopenia. In contrast, sCD4 levels fluctuated during the period of infection. beta 2-M returned within normal range more rapidly than sCD8 secretion. Our results suggest that T cells are activated during infection, and that it is also possible that the raised levels of these soluble molecules play a role in the impairment of protective immunity.

The significance of serum soluble IL-2 receptor as a marker for active visceral leishmaniasis in Sicilian patients.

Sera from nine Sicilian patients with confirmed visceral leishmaniasis (Leishmania donovani infantum; VL), at the moment of the diagnosis, during the course of the disease and after clinical recovery, were analysed for the concentration of soluble IL-2 receptor (sIL-2R). The results show that sIL-2R is a marker of disease activity, since it is in high concentration at the beginning of infection and returns to the normal range following successful chemotherapy. At the same time of serum analysis for sIL-2R, peripheral blood mononuclear cells (PBMC) of VL patients were stimulated with phytohaemagglutinin (PHA) or antigen and supernatant tested for IL-2 and interferon-gamma (IFN-gamma) production. Data demonstrate that there is an inverse relation between concentration of IL-2 and IFN-gamma in the supernatants and sIL-2R secretion in the sera.

An ELISA method in the diagnosis of typhoid fever.

Anti-S. typhi IgG and IgM antibodies were detected by an ELISA test using whole S. typhi as antigen. Among sera of patients suffering from typhus, 100% were positive for IgG and 78.9% for IgM. In the control group (blood donors and patients with various diseases) positivities were 1.01% for IgG and 1.01% for IgM.

Laboratory diagnosis of boutonneuse fever by enzyme-linked immunosorbent assay.

Little is known about the use of the enzyme-linked immunosorbent assay (ELISA) in the diagnosis of boutonneuse fever, and the reports which do exist have concerned the use of a strain of African origin and a peroxidase-labelled conjugate. We have used, as antigen, a Sicilian strain of Rickettsia conorii recently isolated from a patient with boutonneuse fever and anti-human immunoglobulins G and M labelled with alkaline phosphatase as conjugate. 432 (84.6%) of 500 sera from patients with boutonneuse fever, 4.4% of 384 sera from patients with various other pathologies, and 2.9% of 204 sera from blood donors gave positive reactions. The ELISA was highly sensitive and specific in the diagnosis of boutonneuse fever, and seems to be the test of choice for sero-epidemiological investigations, and when a large number of specimens must be examined. The test is more sensitive than the indirect immunofluorescence test, at least in the early stage of the disease (44.9% vs 21.3% positive respectively), and a long time after an acute attack.

Interaction of uridine with GABA binding sites in cerebellar membranes of the rat.

The effect of uridine, a postulated anticonvulsant agent, on GABA receptors has been investigated. Uridine inhibits [3H]GABA binding to rat cerebellar buffer-washed membranes. Pretreatment of the membranes with Triton X-100 increases the effect of uridine on GABA-binding. The Scatchard analysis reveals that both high and low affinities of GABA for its receptors are affected by 1 mM uridine, while the apparent number of binding sites remains unchanged. The ability of uridine to interact competitively with GABA binding sites, also examined by the Lineweaver-Burk analysis, suggests a possible mechanism of action of this anticonvulsant agent, so including it among those compounds characterized by a GABAergic agonist activity.